Kimihiro Komori

Angiogenic Gene Therapy for Experimental Critical Limb Ischemia Acceleration of Limb Loss by Overexpression of Vascular Endothelial Growth Factor 165 but not of Fibroblast Growth Factor-2

Recent studies suggest the possible therapeutic effect of intramuscular vascular endothelial growth factor (VEGF) gene transfer in individuals with critical limb ischemia. Little information, however, is available regarding (1) the required expression level of VEGF for therapeutic effect, (2) the related expression of endogenous angiogenic factors, including fibroblast growth factor-2 (FGF-2), and (3) the related adverse effects due to overexpression of VEGF. To address these issues, we tested effects of overexpression of VEGF165 using recombinant Sendai virus (SeV), as directly compared with FGF-2 gene transfer. Intramuscular injection of SeV strongly boosted FGF-2, resulting in significant therapeutic effects for limb salvage with increased blood perfusion associated with enhanced endogenous VEGF expression in murine models of critical limb ischemia. In contrast, VEGF165 overexpression, 5-times higher than that of baseline on day 1, also strongly evoked endogenous VEGF in muscles, resulting in an accelerated limb amputation without recovery of blood perfusion. Interestingly, viable skeletal muscles of either VEGF165- or FGF-2–treated ischemic limbs showed similar platelet-endothelial cell adhesion molecule-1–positive vessel densities. Maturation of newly formed vessels suggested by smooth muscle cell actin–positive cell lining, however, was significantly disturbed in muscles with VEGF. Further, therapeutic effects of FGF-2 were completely diminished by anti-VEGF neutralizing antibody in vivo, thus indicating that endogenous VEGF does contribute to the effect of FGF-2. These results suggest that VEGF is necessary, but should be delicately regulated to lower expression to treat ischemic limb. The therapeutic effect of FGF-2, associated with the harmonized angiogenic effects seen with endogenous VEGF, provides important insights into therapeutic angiogenesis.

Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits

Background—Renarrowing of dilated arterial sites (restenosis) hampers the clinical benefits of coronary angioplasty. Infiltration and activation of monocytes in the arterial wall mediated by monocyte chemoattractant protein-1 (MCP-1) might be a major cause of restenosis after angioplasty. However, there is no direct evidence to support a definite role of MCP-1 in the development of restenosis. Methods and Results—We recently devised a new strategy for anti–MCP-1 gene therapy by transfecting an N-terminal deletion mutant of the MCP-1 gene into skeletal muscles. We used this strategy to investigate the role of MCP-1 in the development of restenotic changes after balloon injury in the carotid artery in hypercholesterolemic rabbits. Intramuscular transfection of the mutant MCP-1 gene suppressed monocyte infiltration/activation in the injured arterial wall and thus attenuated the development of neointimal hyperplasia and negative remodeling. Conclusions—MCP-1–mediated monocyte infiltration is necessary in the development of restenotic changes to balloon injury in hypercholesterolemic rabbits. This strategy may be a useful and practical form of gene therapy against human restenosis.

Electrical responses of smooth muscle cells during cholinergic vasodilation in the rabbit saphenous artery

Isolated smooth muscle tissues of the rabbit saphenous artery precontracted with norepinephrine (NE) were relaxed by acetylcholine (ACh, greater than 10-7 M) or oxotremorine (greater than 10-7 M), through the activation of muscarinic receptors, only when the endothelial cells were intact. ACh (greater than 10-7 M) transiently hyperpolarized the membrane (1–4 minutes) with an associated decrease in the membrane resistance, either in the presence or absence of NE, and these changes ceased during the continuous application of ACh. The ACh-induced transient hyperpolarization was not generated after mechanically removing the endothelium or by treatment with atropine. Oxotremorine (up to 10-5 M) did not alter the membrane potential in the presence or absence of the endothelium. NE (10-6 M) depolarized the smooth muscle membrane, which remained unchanged by additional application of ACh or oxotremorine for more than 5 minutes, or after removal of the endothelium. The excitatory junction potential generated by perivascular nerve stimulation was inhibited by ACh (greater than 10-9 M) or oxotremorine (greater than 10-9 M) in a concentration-dependent manner. These inhibitory actions of ACh or oxotremorine were blocked by atropine but were not affected by removal of the endothelial cells. These results suggest that the inhibitory actions of muscarinic agonists on electrical responses of smooth muscle cells of the rabbit saphenous artery were mainly indirect, i.e., a release of inhibitory substances from the endothelial cells and the inhibition of adrenerglc transmission. The former required higher concentrations of ACh or oxotremorine, thereby suggesting that the latter may be more important for vasodilation related to cholinergic mechanisms.

Endothelium-Derived Hyperpolarizing Factor

1. Not all endothelium-dependent relaxations can be fully explained by the release of either nitric oxide (NO) and/or prostacyclin. Another unidentified substance(s) that hyperpolarizes the underlying vascular smooth muscle cells (endothelium-derived hyperpolarizing factor; EDHF) contributes to endothelium-dependent relaxations. 2. In blood vessels from various species these hyperpolarizations are resistant to inhibitors of NO synthase (NOS) and cyclo-oxygenase. In canine, porcine and human blood vessels the hyperpolarization cannot be mimicked by nitrovasodilators or exogeneous NO. However, in other species (rat, guinea-pig, rabbit) endothelium-dependent hyperpolarizations resistant to inhibitors of NOS and cyclo-oxygenase and hyperpolarizations to endothelium-derived or exogeneous NO can be observed in the same vascular smooth muscle cells. 3. In blood vessels where NO causes hyperpolarization, the response is blocked by glibenclamide, suggesting the involvement of ATP-dependent potassium channels. Hyperpolarizations caused by EDHF are insensitive to glibenclamide but, depending on the tissue, are inhibited by relatively small concentrations of tetraethylammonium (TEA) or by apamin or the combination of charybdotoxin plus apamin, indicating that calcium-dependent potassium channels are likely to be involved. 4. Metabolites of arachidonic acid, through the cytochrome P450 mono-oxygenase pathway (epoxyeicosatrienoic acids), are produced by the endothelial cells, increase the open-state probability of calcium-activated potassium channels sensitive to TEA or charybdotoxin, and induce the hyperpolarization of arterial smooth muscle cells, indicating that epoxyeicosatrienoic acids could be EDHF. However, in blood vessels from various species, cytochrome P450 inhibitors do not affect endothelium-dependent hyperpolarizations, indicating that EDHF is not yet identified with certainty. 5. Endothelium-derived hyperpolarizing factor released from cultured endothelial cells reduces the intracellular calcium concentration in vascular smooth muscle cells and the EDHF component of the relaxation is proportionally more important in smaller than larger arteries. In aging animals and in various models of diseases, endothelium-dependent hyperpolarizations are diminished. 6. The identification of EDHF and/or the discovery of specific inhibitors of its synthesis and its action may allow a better understanding of its physiological and pathophysiological role(s).

Heterogeneous distribution of muscarinic receptors in the rabbit saphenous artery

1 The properties of the muscarinic receptors in the rabbit saphenous artery were determined from electrical and mechanical responses of smooth muscle cells produced by acetylcholine (ACh). The inhibitory action of atropine and pirenzepine on the ACh‐induced responses was also studied. 2 ACh produced a transient hyperpolarization of the membrane and inhibited the noradrenaline (NA)‐induced contraction. These effects of ACh were apparent only when the endothelial cells were intact. 3 The ACh‐induced transient hyperpolarization was antagonized by atropine or pirenzepine, with similar potencies (the ID50 values were about 2 × 10−8 m for both antagonists). 4 The ACh‐induced inhibition of the contraction to NA was antagonized by atropine more preferentially than by pirenzepine (the ID50 values were 2 × 10−8 m for atropine and 10−6 m for pirenzepine). 5 The excitatory junction potential (e.j.p.) evoked by perivascular nerve stimulation was inhibited by ACh (above 10−8 m). The ACh‐induced inhibition of the e.j.p. was antagonized by atropine more preferentially than by pirenzepine (the ID50 values were 3 × 10−8 m for atropine and 6 × 10−6 m for pirenzepine). 6 It is concluded that in the rabbit saphenous artery, two subtypes of muscarinic receptor (M1 and M2) are located on the endothelial cells. Stimulation of each subtype releases a different substance, i.e., a hyperpolarizing substance (M1‐subtype) or a relaxant substance (M2‐subtype). In prejunctional nerve terminals, the muscarinic receptors responsible for inhibiting the release of transmitter substances are of the M2‐subtype.

Detection of active cytomegalovirus infection in inflammatory aortic aneurysms with RNA polymerase chain reaction

PURPOSE We previously reported the possible role of human cytomegalovirus in the pathogenesis of inflammatory aortic diseases. To further analyze the viral cause of human aortic diseases, in this study we examined the presence and the replication of human Herpesviridae in 60 aortic tissues, including 7 inflammatory aneurysms, 37 atherosclerotic aneurysms, and 16 normal aortas. METHODS To detect the genome of herpes simplex virus (type 1, type 2), cytomegalovirus, and Epstein-Barr virus, DNA polymerase chain reaction for each virus was performed. To analyze these herpesviral replications, the viral transcript was detected with RNA polymerase chain reaction. RESULTS The DNA polymerase chain reaction showed that either herpes simplex virus or cytomegalovirus was present more frequently in inflammatory (29% or 86%, respectively) and atherosclerotic aneurysms (27% or 65%, respectively) than in normal aortic tissues (6% or 31%, respectively), whereas the Epstein-Barr viral genome was not detected in any aortic tissue specimens. By the use of RNA polymerase chain reaction, only the cytomegaloviral transcript was recognized in 71% of the inflammatory aneurysms but was not recognized in any other tissue specimens. No other herpesviral transcripts were detected in any tissue specimens examined in this study. CONCLUSIONS Our results thus suggest that the human herpesviruses may play various roles in the pathogenicity of aortic diseases, in particular the replicating infections of the cytomegalovirus might potentially cause the formation of inflammatory aneurysms.

Recombinant Sendai virus-mediated gene transfer to vasculature: a new class of efficient gene transfer vector to the vascular system

Unsolved issues in clinical gene therapy for cardiovascular diseases include gene transfer efficiency and the requirement of a longer exposure time. We developed a novel mononegavirus vector, recombinant Sendai virus (SeV), and tested whether it can overcome the present hurdles. SeV showed dose‐dependent and persistent gene expression in either proliferating or arrested cells, suggesting stability of RNA genome of the vector. An outstanding feature of the SeV‐mediated gene transfer was that even a brief exposure provided nearly peak gene expression in both culture cells and human veins ex vivo, as well as rabbit carotid arteries in vivo. Gene transfer to human great saphenous veins showed high efficacy in luminal and vasa vasoral endothelial cells and in adventitial fibroblasts via both intraluminal delivery and simple floating; however, only scattered cells were transfected in both neointima and media, regardless of the infusion pressure. Veins with a dissected neointima showed a clear transfection to medial cells, suggesting that the barrier in neointima reduces SeV‐mediated gene transfer to tunica media, similar to the case with adenoviruses. Although the fibromuscular neointima is a common obstacle, these findings suggest that SeV may overcome other limitations of current vectors. SeV may be an important new vector in treating subjects with vascular disorders.

Molecular Evaluation of Endothelial Progenitor Cells in Patients With Ischemic Limbs Therapeutic Effect by Stem Cell Transplantation

Objective—Although some patients with limb ischemia have recently undergone therapeutic angiogenesis by cell transplantation, their angiogenic potential has not been well characterized. It is also important to evaluate endothelial progenitor cell (EPC) contents in different stem cell sources to choose the best material for therapeutic angiogenesis. Methods and Results—We quantitated the mRNA expression of EPC-specific molecules (eg, Flk-1, Flt-1, CD133, VE-cadherin, etc) in bone marrow-derived or peripheral blood-derived mononuclear cells obtained from patients with ischemic limbs, using real-time reverse-transcription polymerase chain reaction technique. The mRNA expression level of EPC markers was significantly lower in the patients than in healthy controls, which was consistent with results of flow cytometric analysis. However, the implantation of autologous bone marrow mononuclear cells increased the circulating EPCs in the peripheral blood of patients. We furthermore revealed the different expression pattern of EPC markers in possible sources for stem cell transplantation, including normal bone marrow, peripheral blood obtained from recombinant granulocyte colony–stimulating factor-treated donor, and umbilical cord blood. Conclusions—Patients with peripheral obstructive arterial diseases may have lower angiogenic potential because of decreased expression of EPC specific molecules in their marrow and blood. Therapeutic angiogenesis by transplantation of autologous marrow mononuclear cells increased circulating EPCs in the patients and improved ischemic symptoms.

Does endothelin-1 mediate endothelium-dependent contractions during anoxia?

Summary Endothelin-1 (ET-1) is a more potent constrictor of femoral venous than femoral arterial smooth muscle. By contrast, endothelium-dependent contractions to anoxia are more prominent in the artery. Unlike those to ET-1, the endothelium-dependent contractions evoked by anoxia are rapid in onset and readily reversible; they are antagonized by Ca2+ entry blockers. ET-1-induced responses are inhibited by endothelium-dependent relaxing factors in canine arteries, and to a lower extent in veins. ET-1 does not augment the production of cyclic GMP in cultured porcine aortic endothelial cells, or in canine arteries and veins, suggesting that the peptide does not evoke the release of endothelium-dependent relaxing factor (EDRF) in canine blood vessels. The endothelium-derived contracting factor (EDCF) released during anoxic contraction cannot be bioassayed, under conditions where ET-1 causes contraction of the bioassay tissue. No ET-1 appears to be released in the extracellular space during anoxic contractions. These findings do not support the hypothesis that ET-1 is the EDCF released by anoxia.

Endothelium-dependent vasorelaxations in response to aggregating platelets are impaired in reversed vein grafts

The endothelium releases factor(s) that are potent vasodilators and inhibitors of platelet aggregation. Experiments were performed to determine whether the endothelium-dependent responses to aggregating platelets are altered in vein grafts. Segments of jugular veins were grafted in the reverse position into the carotid arteries in 16 rabbits. After 4 weeks the patent grafts (14 of 16) were removed, and the endothelium-dependent responses were examined in vitro. In control veins aggregating platelets, adenosine diphosphate, and serotonin caused endothelium-dependent relaxations. The platelet-induced relaxations were attenuated by apyrase (adenosine diphosphatase and adenosine triphosphatase) but not by methiothepin (serotonergic blocker). In vein grafts, endothelium-dependent relaxations in response to aggregating platelets were absent, and only contractions that could be attenuated by methiothepin were observed. In vein grafts, endothelium-dependent relaxations in response to adenosine diphosphate were reduced, and only endothelium-independent contractions were observed in response to serotonin. These contractions were attenuated by methiothepin. These results suggest that (1) the endothelium exerts an inhibitory effects mediated mainly by adenosine diphosphate in response to aggregating platelets in rabbit jugular veins and (2) endothelium-dependent relaxations in response to aggregating platelets are impaired in vein grafts because of reduced endothelium-independent contractions in response to serotonin. This impairment of endothelium-dependent responses in vein grafts may contribute to failure of the grafts.